- Title
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Cell adhesion in zebrafish embryos is modulated by march8
- Authors
- Kim, M.H., Rebbert, M.L., Ro, H., Won, M., Dawid, I.B.
- Source
- Full text @ PLoS One
(A) March8 MO-injected embryos showed different levels of morphological defects as compared with WT, including cell death (arrowhead), shortened body axis and kinked tail (arrow). (B) Quantitative evaluation of defects in embryos injected with 10 ng March8 MO. These defects were rescued by coinjection with 10 pg march8 mRNA. Number of embryos is listed on top of bars. PHENOTYPE:
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(A–C) Uninjected embryos (A), and embryos injected with WT march8 mRNA (B) or RING domain mutant (W109A) march8 mRNA (C), at shield to 60% epiboly stage. Overexpression of March8 results in loss of cell adhesion, abnormal cell migration, and cell death (B); individual embryos showing disaggregation of embryonic cells are shown in B2. Embryos injected with W109A mutant mRNA were normal (C). (D) Self ubiquitination of March8. HEK293T cells were transfected with the plasmids indicated and activated by addition of tebufenozide (see Materials and Methods). Lysates were immunoprecipitated (IP) with anti-March8 antibody and then immunoblotted (IB) with anti-HA antibody to detect ubiquitinated proteins (upper panel). Expression of March8 and tubulin as control are shown in bottom panels. |
The RING domain of March8 is critical for induction of embryonic defects. The classification of morphological defects at 26 hpf after march8 mRNA injection is shown at the left. Quantification shows that defects were induced in a dose-dependent manner by WT, but very weakly by W109A mutant march8 mRNA. Number of embryos is listed on top of bars. UI, uninjected; GFP, embryos injected with 50 pg GFP mRNA. |
March8 down-regulates cell surface levels of E-cadherin. Control and march8 mRNA (25 pg) injected embryos were fixed at germ ring stage and stained with anti-March8 (red) and anti-E-cadherin (green) antibodies. Confocal images of EVL and deep cells are shown in animal pole view. March8 overexpression is visualized by higher levels of March8 in intracellular vesicles, and leads to reduced cell surface localization of E-cadherin. EXPRESSION / LABELING:
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Expression pattern of march8 during embryogenesis. (A) Total RNA was extracted from staged embryos, and march8 mRNA was analyzed by RT-PCR; β-actin was used as a loading control. (B) March8 mRNA expression in zebrafish embryos was detected by in situ hybridization. Shown are 2 cell, 70% epiboly and tailbud stage; lateral views with animal pole on top. Three and 10 somite; left: lateral view with dorsal to the right; right: dorsal view anterior to the top. One and two day embryos; lateral view with anterior to the left. |
March8 MO injection increases apoptosis in zebrafish embryos. Detection of apoptotic cells by TUNEL assay in zebrafish embryos at 26 hpf (lateral views). Uninjected embryos showed a low level of apoptotic cells. March8 MO injection increased apoptotic cells in the entire embryo. PHENOTYPE:
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